Calmodulin-stimulated protein kinase activity from rat pancreas.

Abstract

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein $6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When *251-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calciumcalmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.Neurohumoral stimulation of the exocrine pancreas by agents such as carbachol and cholecystokinin results in alterations in protein phosphorylation (1-4) and intracellular calcium levels (5, 6). Recent studies from this laboratory have demonstrated that an Mr 29,000 ribosomal protein which has been putatively identified as $6 in pancreas undergoes phosphorylation in close association with secretagogue action. This phosphorylation is stimulated in situ in 32Pi-labeled pancreatic lobules by carbachol and cholecystokinin and in pancreatic homogenates by calcium and cAMP using [-r-32p]ATP as the phosphate donor (2-4). In this report we describe a calciumcalmodulin-dependent protein kinase (CaM-PK ~) in rat pancreatic postmicrosomal supernatant that phosphorylates the Mr 29,000 ribosomal protein. Partial purification of this CaM-PK activity yielded both an Mr 51,000 calcium-binding protein (CBP) and an Mr 51,000 phosphoprotein which underwent phosphorylation in a calcium-calmodulin-dependent manner. This CaM-PK activity resembled calmodulin-regulated kinases that have recently been characterized in liver Abbreviations used in this paper: CaM-PK, calcium-calmodulindependent protein kinase; CBP, calmodulin-binding protein.using glycogen synthase as substrate (7,8) and in brain using synapsin I (9) and tubulin (10) as substrates. MATERIALS AND METHODSTissue Preparation: Pancreata were obtained from fed, male, Sprague-Dawley rats ( 150-200 g) following decapitation. The pancreata were homogenized in 0.3 M sucrose contai...

Rabbit ileal villus cell brush border Na+/H+ exchange is regulated by Ca2+/calmodulin-dependent protein kinase II, a brush border membrane protein.

“…A number of isozymes of the CaM kinase II exist with variable ratios ofthe subunits among tissues. For example, the enzyme derived from some parts of the brain contains a minor 52-kDa subunit, that isolated from the liver contains a doublet of 52 and 54 kDa, while the enzyme from pancreas contains a single 52-kDa subunit (24). The CaM kinase II in broken cell studies in which access to the transport protein regulated was not an issue.…”

Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme.

“…The kinase phosphorylates synapsin I (28) and tyrosine hydroxylase (4) in situ. Many other proteins, including microtubule-associated protein 2 (MAP-2), r proteins, tryptophan 5'-monooxygenase, ribosomal protein S6, vimentin, and myosin light chains, are good substrates in vitro and are candidates for physiological substrates (3,6,13). Therefore, the Ca2+/calmodulin-dependent protein kinase may be involved in the regulation and coordination of numerous cellular processes.…”

Effects of Calmodulin Inhibitors on Amylase Secretion from Rat Pancreatic Acini

“…Calmodulin has been reported to be responsible for activating several kinases, phosphatase and cyclic nucleotide phospho diesterase in the pancreas [4], A Ca2+-calmodulin-dcpendent protein kinase (CaM kinase) was partially purified from rat pancreas, which phosphorylated a ribosomal protein with a molecular weight (Mr) of 29.000 [5]. This CaM kinase fraction contained a phosphoprotein of Mr 51.000 whose phosphoryla tion was dependent on Ca2+ and calmodulin.…”